Antibody/receptor targeting moiety for enhanced delivery of armed ligand

ABSTRACT

A method for intracellular delivery of drugs or other agents for diagnosis and therapy of malignancies or immune-mediated or inflammatory conditions. A targeting moiety of a an antibody and the ligand-binding region of a selected cytokine receptor is used. The targeting moiety targets surface antigen on a specific cell population. The targeting moiety is administered to a subject, and then, after a specified interval, therapeutic or diagnostic agents linked to the cognate cytokine then are given. The invention provides rapid, efficient internalization of the cytokine receptor antibody/antigen complexes. Targeting of a high-level cell surface antigen with such bispecific fusion molecules substantially increases the number of cytokine receptors over their low background level.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/231,642, filed Jan. 15, 1999, which claims benefit from U.S.Provisional Application No. 60/071,520, filed Jan. 15, 1998, both ofwhich are incorporated herein by reference in their entirety.

GOVERNMENT LICENSE RIGHTS

[0002] The U.S. Governrnent has a paid-up license in this invention andthe right in limited circumstances to require the patent owner tolicense others on reasonable terms as provided for by the terms of grantnumbers CA 39841-13 and RR 12603, awarded by the National Institutes ofHealth.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to a conjugate, preferably a fusionprotein, of a component of an internalizing receptor complex and amonoclonal antibody (mAb) that binds to a specific surface antigen on acell, to a conjugate of a radionuclide or toxin and a ligand for theinternalizing receptor system, and to a method of diagnosis or therapyusing such genetic or chemical conjugates.

[0004] There is now a fairly large and growing body of experience in theuse of mAbs for tumor therapy. Several studies targeting differentantigens have shown promising results. These studies have usedradiolabeled mAbs and, to a lesser extent, mAb-toxin conjugates.

[0005] mAbs used in tumor diagnosis and therapy differ in their abilityto bind cognate antigen and to become internalized. For example, CD22exhibits efficient internalization as well as reexpression of thisantigen after internalization. It suffers, however, from relatively lowexpression levels on some B-cell malignancies, e.g., it is expressed ononly 30-50% of cases of B-cell lymphocytic leukemia (B-CLL).

[0006] Other cell surface antigens such as HLA-DR and the CD20 antigen,in contrast to the CD22 antigen, are quite highly expressed B-cellantigens that are expressed on a wide range of B-cell malignancies,ranging from acute lymphocytic leukemia (ALL) to the more differentiatedB-Cell (B-CLL) and non-Hodgkin's lymphoma (NHL), and even to hairy cellleukemia (HCL). These antigens are generally expressed on cells in thevast majority of cases of these malignancies at a high antigen density.A major disadvantage of these antigens is that they slowlyinternalizing. This feature militates significantly against targetingHLA-DR and CD20 for toxin-based therapy.

[0007] A further problem of HLA-DR and CD20 is the fact that B-cellmalignancies exhibit a more rapid dissociation of bound anti-HLA-DR andanti-CD20 mAbs from the surface as compared to nonlymphoma tumor cells.This suggests that a therapy that targets a B-cell restricted antigen,particularly those characterized by slow internalization, could beenhanced by addressing these issues.

[0008] A variety of mAb-toxin constructs have been tested in both invitro experiments and human trials. These studies have demonstratedpotent and specific effects of these reagents. Most of the toxinmolecules that have been used derive from either plant or bacterialsources and hence produce neutralizing anti-toxin antibody responses inpatients. This severely limits the duration of therapy.

SUMMARY OF THE INVENTION

[0009] It is therefore an object of the present invention to providemore effective methods of diagnosis and/or therapy for cancer andimmunologically-mediated or infectious diseases.

[0010] It is another object of the invention to improve the value asantigenic targets of slowly internalizing surface antigens.

[0011] It is a further object of the invention to reduce the tendency ofantibodies bound to the surface of tumor cells to dissociate from thesurface of the cells.

[0012] These and other objects of the invention are achieved byproviding a targeting moiety comprising a conjugate of an antibodylinked to a ligand-binding region of a receptor subunit selected fromthe group consisting of IL-2Rα, IL-4Rα, IL-13Rα and IL-15Rα, whichantibody is specific for a cellular antigen specific to a targeted cell.The targeting moiety may comprise a covalent conjugate in which theantibody is covalently linked to the ligand-binding region, a fusionprotein of the antibody and the ligand-binding region, or a bispecificantibody that has a first specificity for a cellular antigen specific toa targeted cell and a second specificity for a rapidly internatlizingreceptor complex. In one embodiment, the antibody is specific to anantigen expressed by solid tumors, for example, CEA, and is linked tothe ligand-binding region of IL-13Rα. In an alternative embodiment, theantibody is specific to HLA-DR and is linked to the ligand-bindingregion of IL-15Rα. A composition comprising a targeting moiety accordingto the invention and a pharmaceutically acceptable carrier also isprovided.

[0013] A kit comprising a conjugate of IL-13 linked to a drug,radionuclide or toxin, and a targeting moiety comprising an antibodyspecific for a cell marker specific to a targeted cell, linked to theligand-binding region of IL-13Rα, is provided. A second kit comprising aconjugate of IL-15 linked to a drug, radionuclide or toxin, and atargeting moiety comprising an antibody specific for a cell markerspecific to a targeted cell, linked to the ligand-binding region ofIL-15Rα, also is provided.

[0014] The invention provides a method of treatment for cancer,comprising first administering to a subject in need of such treatment atargeting moiety comprising an antibody specific for an antigen specificto a targeted cell, linked to the ligand-binding region of IL-13Rα, andthen, after a predetermined time interval, administering to the subjecta therapeutically effective amount of a conjugate of IL-13 linked to adrug, radionuclide or toxin. Another method of treatment for cancer oran immunologically-mediated or infectious disease comprises firstadministering to a subject in need of such treatment a targeting moietycomprising an antibody specific for an antigen specific to a targetedcell, linked to the ligand-binding region of IL-15Rα, and thenadministering to the subject a therapeutically effective amount of aconjugate of IL-15 linked to a drug, radionuclide or toxin.

[0015] Other objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0016] It has been discovered, surprisingly, that the value of surfaceantigens as antigenic targets can be improved significantly byfunctionally linking them to a high affinity, internalizing receptorsystem. The present invention is of particular advantage in the case ofuseful cell surface antigens that internalize slowly.

[0017] The present invention is based on a fundamental property ofcytokine and growth factor receptors, viz., their ability to rapidly andefficiently internalize. Examples of rapid internalization of receptorand ligand include intracellular transport of nutrients, as with thetransferrin and low-density lipoprotein (LDL) receptors. Receptors forgrowth factors like insulin and epidermal growth factor (EGF) as well ascytokine receptors such as IL-1R, IL-2R and IL-4R also internalizerapidly. In all cases studied, except that of transferrin, the ligandundergoes proteolysis as a consequence of trafficking to the low pH,protease/acid hydrolase-containing lysosomal compartment. Moleculesassociated with the ligands, such as cholesterol bound to LDL or drugs,toxins, and radionuclides, linked to other ligands can exit toextra-lysosomal compartments where they can exert their effects.

[0018] The fate of a receptor following internalization varies dependingon the receptor system. For example, it may recycle to the cell surface,as with the transferrin and LDL receptors, or it may itself be degraded.Lysosomal degradation of receptors has been reported for receptors suchas the EGF receptor and contributes to receptor down-regulation anddesensitization to subsequent ligand stimulation.

[0019] In some ligand/receptor systems, there is re-expression ofreceptors via de novo mRNA and protein synthesis. For example, CD22 isinternalized rapidly after binding of the cognate LL2 mAb and isre-expressed as soon as 2 hours after a complete cycle of antigensaturation binding of cognate antigen by the specific mAb followed byinternalization at 37° C. Further evidence of re-expression is found inthe ability of a wide array of cytokine/growth factor-dependent celllines to be maintained for months or even years, suggesting ongoingre-synthesis and re-expression of the requisite ligand binding as wellas the associated signaling proteins. Taken together, these observationsshow that cytokine receptors are capable of multiple, rapid cycles ofinternalization and re-expression and hence have a high capacity forintracellular delivery of ligands.

[0020] In accordance with the present invention, it is possible toinduce rapid internalization of a slowly internalizing antigen bybringing it in juxtaposition with a more rapidly internalizing complex.For example, the IL-2 receptor system consists of an alpha (IL-2Rα,formerly Tac antigen), beta (IL-2Rβ) and gamma (γ_(c)) chain. IL-2Rαinternalizes slowly, but once it becomes physically associated to IL-2Rβand γ_(c) by the presence of the IL-2 ligand the entire trimeric proteincomplex becomes internalized at the rapid intrinsic rate of theIL-2Rβ/γ_(c) dimer. This follows a recurring pattern in cytokinebiochemistry, in which a functional receptor consists of two or moresubunits, one of which is typically a private, specific alpha chain. TheIL-6-IL-6 system is particularly notable in that the extracellulardomain of the alpha subunit has an intrinsic ability to associate withthe gp130 signaling molecule such that, when IL-6 plus a soluble form ofIL-6Rα are added to cells that express only gp130, a signaling responseoccurs. The IL-2 and IL-6 receptor systems are exemplary of two majorcytokine signaling subunits, γ_(c) (utilized by receptor complexes forILs-2, 4, 7, 9 and 15) and gp130 (utilized by receptor complexes forILs-6 and 11, CNTF, LIF, OSM and cardiotrophin-1), respectively.

[0021] Receptor systems can be harnessed according to the presentinvention to provide enhanced intracellular delivery of armed ligands.Cytokine receptors can be targeted to the surface of cells that normallylack such receptors by the use of mAb-receptor conjugates. For example,the alpha chains of the IL-6 and the ciliary neurotrophic factor (CNTF)receptors have been targeted to the surface of previously negative cellsby way of mAbs directed against the CD34, CD45 and CD64 cell surfaceantigens. Addition of such mAb-Rα conjugates to factor-dependent cellsconferred de novo, specific responsiveness to IL-6 or CNTF.

[0022] A targeting moiety according to the invention comprises areceptor linked to a mAb fragment up to F(ab′)₂ size. Suitable antibodyfragments include F(ab′)₂, F(ab)₂, Fab′, Fab, Fv and the like, includinghybrid fragments. Also useful are any subfragments that retain thehypervariable, antigen-binding region of an immunoglobulin. This willinclude genetically engineered and/or recombinant proteins, whethersingle-chain or multiple-chain, which incorporate an antigen bindingsite and otherwise function in vivo as targeting vehicles insubstantially the same way as natural immunoglobulin fragments.Single-chain binding molecules are disclosed in U.S. Pat. No. 4,946,778,which is hereby incorporated by reference. Fab′ antibody fragments maybe conveniently made by reductive cleavage of F(ab′)₂ fragments, whichthemselves may be made by pepsin digestion of intact immunoglobulin. Fabantibody fragments may be made by papain digestion of intactimmunoglobulin, under reducing conditions, or by cleavage of F(ab)₂fragments which result from careful papain digestion of whole Ig. Thefragments may also be produced by genetic engineering. When the term“antibody” is used herein, all the above types of fragments are includedtherein.

[0023] mAb fragments such as the single chain antibody scF_(v) have theadded advantages of rapid blood and organ clearance and improvedpenetration into tumor nodules and, in a preferred embodiment, scF_(v)of a mAb to a desired target antigen is linked to the ligand-bindingregion of a receptor. mAb molecular engineering techniques can be usedto produce scF_(v). This molecule can be produced by cloning the V_(H)and V_(L) segments from the mAb of interest and splicing them togetherwith a short linker region interposed between them. These molecules,after proper design and renaturation, retain the antigen bindingactivity of the parent mAb and can be expressed at high levels in E.coli-based insect or mammalian expression systems. These constructs thencan provide a platform for the engineering of bifunctional single chainmolecules that can link a second moiety (receptor or a single chainantibody) to the first to retarget effector cells or molecules.

[0024] Mixtures of antibodies, as well as hybrid antibodies, can beused. The hybrids can have two specificities, e.g., one arm binding toone antigen on the target cell and another arm binding to anotherantigen on the target, or one arm could possess a ligand binding regionof a receptor subunit. Hybrid antibody fragments with dual specificitiescan be prepared analogously to the anti-tumor marker hybrids disclosedin U.S. Pat. No. 4,361,544. Other techniques for preparing hybridantibodies are disclosed in, e.g., U.S. Pat. Nos. 4,479,895, 4,474,893,4,714,681, and in Milstein et al., Immunol. Today 5:299 (1984), allincorporated herein by reference. The foregoing are merely illustrative,and other combinations of specificities can be envisioned that also fallwithin the scope of the invention.

[0025] The antibody is linked to the extracellular domain of a receptor.Since the entire extracellular domain is large, truncated versions ofthe domain that contain the ligand-binding site can be used. Theantibody/receptor conjugate can be formed by covalently linking theantibody to the receptor, directly or through a short or long linkermoiety, through one or more functional groups on the anti-body and/orthe enzyme, e.g., amine, carboxyl, phenolic, thiol or hydroxyl groups,to form a covalent conjugate. Various conventional linkers can be used,e.g., diisocyanates, diisothiocyanates, bis(hydroxysuccinimide) esters,carbodiimides, bismaleimides, dithiols, maleimide-hydroxysuccinimideesters, glutaraldehyde and the like. The antibody construct may bind onearm to either the ligand binding region or a site that is remote fromthe ligand-binding site depending on whether ligand will be employed ina given application.

[0026] A simple method is to mix the antibody with the ligand-bindingregion in the presence of glutaraldehyde to form the antibody/receptorconjugate. The initial Schiff base linkages can be stabilized, e.g., byborohydride reduction to secondary amines. This method is conventionallyused to prepare, e.g., peroxidase-antibody conjugates forimmunohistochemical uses or for immunoassays. A diisothiocyanate, abishydroxysuccinimide ester, carbodiimide or other biofunctionalcrosslinkers can be used in place of glutaraldehyde.

[0027] More selective linkage can be achieved by using aheterobifunctional linker such as a maleimide-hydroxysuccinimide ester.Reaction of the latter with the ligand-binding region of the receptorwill derivatize amine groups on the receptor, and the derivative canthen be reacted with, e.g., an antibody Fab fragment with freesulfhydryl groups (or a larger fragment with sulfhydryl groups appendedthereto by, e.g., Traut's Reagent).

[0028] It is advantageous to link the ligand-binding region of thereceptor subunit to a site on the antibody remote from the antigenbinding site. This can be accomplished by, e.g., linkage to cleavedinterchain sulfhydryl groups, as noted above. Another method involvesreacting an antibody whose carbohydrate portion has been oxidized, witha ligand-binding region which has at least one free primary amino group.This results in an initial Schiff base (imine) linkage, which ispreferably stabilized by reduction to a secondary amine, e.g., byborohydride reduction, to form the final conjugate.

[0029] Alternatively, the antibody/receptor conjugate comprises abispecific antibody conjugate which is linked immunologically to theligand-binding region of a receptor. The bispecific antibody or antibodyfragment has a first specificity for a cellular antigen specific to atargeted cell and a second specificity for an extracellular antigenspecific to a targeted cell and a second specificity for anextracellular retion of the ligand-binding subunit or another subunit ofthe receptor complex.

[0030] Most preferably, the antibody/receptor conjugate comprises afusion protein, in which a fusion sequence comprising antibody linked toa ligand-binding region of the receptor is expressed in a recombinantvirion-based, mammalian expression system or other mammalian, insect,yeast or E. coli-based expression system. Suitable linkers for linkingthe antibody to the ligand-binding region are, for example, (GGSBS)₃(SEQ ID NO: 1) or the 23-amino acid linker disclosed in Kurucz et al., JImmunol. 154: 4576-4582 (1995).

[0031] Any of the antibody/receptor conjugates can be labeled with, orconjugated or adapted for conjugation to, a radioisotope or magneticresonance image enhancing agent. In a preferred embodiment, theantibody/receptor bifunctional construct is unlabeled. It isadministered and then, after a predetermined interval sufficient forlocalization to the target site and also clearance from the circulatorysystem of the mammal, the armed cognate ligand will be given. This isparticularly important when boron complexes are used therapeutically.Alternatively, the conjugate is tagged with a label, e.g., a radiolabel,a fluorescent label or the like, that permits its detection andquantitation in body fluids, e.g., blood and urine, so that targetingand/or clearance can be measured and/or inferred.

[0032] Any conventional method of radiolabeling which is suitable forlabeling proteins for in vivo use, generally is suitable for labelingthese antibody/receptor conjugates. This can be achieved by directlabeling with, e.g., I-131, I-124 or I-123. Labeling with either I-131,I-124 or I-123, is readily effected using an oxidative procedure whereina mixture of radioactive potassium or sodium iodide and the antibody istreated with chloramine-T, e.g., as reported by Greenwood et al.,Biochem. J, 89: 114 (1963) and modified by McConahey et al., Int. Arch.Allergy Appl. Immunol., 29: 185 (1969). This results in directsubstitution of iodine atoms for hydrogen atoms on the antibodymolecule, presumably mostly on tyrosine residues, possibly also ontryptophan and even on phenylalanine residues, depending on theproportions of reagents and the reaction conditions. Alternatively,either lodogen-based methods as described by Fraker et al., BiochemBiophys Res. Commun 80:849-857, 1978, or lactoperoxidase iodination maybe used, as described by Feteanu, supra, page 303, and references citedtherein.

[0033] Instead of I-131, I-124 or I-123, the conjugate can be labeled bymetallation with, e.g., Tc-99m or Cu ions or the like, by conventionaltechniques, or by attaching a chelator for a radiometal or paramagneticion. Such chelators and their modes of attachment to antibodies are wellknown to the ordinary skilled artisan.

[0034] The antibody/receptor conjugate acts as a targeting moietyaccording to the invention when administered to a patient. This isfollowed by a targeting and clearance interval that allows for bindingof the targeting moiety to intended target cells and its clearance fromnormal tissues, after which armed ligand is administered. The armedligand comprises the cognate ligand for the receptor of the targetingmoiety, conjugated to a radionuclide, drug or toxin. The radionuclidecan be either a diagnostic or therapeutic radionuclide.

[0035] Examples of diagnostic radionuclides include iodine-123,iodine-124, iodine-131, indium-111, gallium-67, gallium-68,ruthenium-97, technetium-94, technetium-99m, copper-64, copper-67,cobalt-57, cobalt-58, chromium-51, iron-59, yttrium-86, selenium-75,thallium-201, and ytterbium-169. Examples of therapeutic radionuclidesinclude alpha-emitters, e.g., bismuth-212 and astatine-211;beta-emitters, e.g., yttrium-90, rhenium-186, rhenium-188, copper-67 andiodine-131; and alternatively, electron capture or Auger conversionelectronemitting radionuclides such as iodine-125, indium-111 andgallium-67. Preferably the radionuclide will emit in the 10-5000 keVrange, more preferably in the 50-1500 keV range, and most preferably inthe 50-500 keV range. The radionuclides may be incorporated into thespecific antibody by the labeling techniques discussed above, as well asother conventional techniques well known to the art.

[0036] Many drugs and toxins are known which have a cytotoxic effect ontumor cells or microorganisms that may afflict humans and mammals ingeneral. They are to be found in compendia of drugs and toxins, such asthe Merck Index and the like. A preferred toxin is a ribonuclease, suchas onconase. Onconase is a non-mammalian RNAse purified from Ranapipiens oocytes. It has been shown in clinical trials to have anti-tumoractivity against human pancreatic cancer, but has been found to haveminimal anti-tumor activity against B-cell malignancies such as B-celllymphoma or leukemia.

[0037] Addition of the armed ligand (specifically IL-2 and IL-15)results in the formation of a trimeric complex comprising the β/γ_(c)chain subunits present on the surface of the targeted cell and theα-chain subunit of receptor, which is attached to the surface of thetargeted cell by means of the antibody/receptor conjugate. This leads torapid internalization of toxin and/or radionuclide into the targetedcells. An analogous dimeric or trimeric complex assembles after theaddition of IL-4 or IL-13. While internalization is not necessary for atherapeutic radionuclide to be effective, these multimeric complexesprovide a tighter, more stable binding of the ligand to the targetedcells, and also facilitates internalizations.

[0038] The same ligand can be armed with both radionuclide, drug andtoxin, or separate ligands can be armed with radionuclide, drug andtoxin. Where separate ligands armed with radionuclide, drug and toxinare used, these may be administered together or sequentially.

[0039] The antibody/receptor conjugate and armed ligand conjugate areadministered in a composition with a pharmaceutically acceptablecarrier. In this regard, a pharmaceutically acceptable carrier is amaterial that can be used as a vehicle for administering the fusionprotein or armed ligand because the material is inert or otherwisemedically acceptable, as well as compatible with the fusion protein orarmed ligand.

[0040] Preferred high affinity, internalizing receptor systems to whichthe antibody can be linked include the IL-2, IL-4, IL-13 and IL-15receptor systems. Internalization rates for IL-2/IL-2R (related to theIL-15/IL-15R system) and for IL-4/IL-4R (related to the IL-13/IL-13Rsystem) have t_(1/2) values of approximately 15-30 minutes. Particularlypreferred receptor systems for use in the present invention are theIL-13 and IL-15 receptor systems.

[0041] The IL-13/IL-13 receptor system is a good candidate foraddressing solid tumors due to its widespread expression and signalingcapability on cancers of epithelial origin (preponderantly CEA+). TheIL-13/IL-13 receptor system shares several features in common with theIL-4/IL-4 receptor system. Structurally, the IL-4 and IL-13 ligands showapproximately 30% protein sequence homology, which is the highestamongst the interleukins. Both receptors possess the canonical fourconserved cysteine residues in the N-terminal half of theirextracellular domains as well as a WSXWS motif in the juxtamembraneregion of their extracellular domains, and are members of thehematopoietin superfamily. IL-13Rα is of smaller overall size thanIL-4Rα, but has a somewhat larger extracellular domain and acorrespondingly shorter intracellular domain.

[0042] IL-13 and IL-4 have considerable functional similarity as well.Both suppress production of pro-inflammatory cytokines by macrophages,are co-stimulatory for B cell proliferation and induce immunoglobulinisotype switching. Both induce upregulation of both CD23 and MHC classII on both monocytes and B cells. IL-13 and IL-4 bind nonhematopoieticcells, such as carcinoma cell lines, with high affinity and exertbiologic effects on them. IL-4 inhibits the growth of these epithelialcancer cell lines in unmodified form both in vitro and in vivo.Unmodified IL-13 also has in vitro growth-inhibitory effects on breastcarcinoma cell lines, and thus shares this property.

[0043] In several cell types, IL-13 competes for IL-4 binding and viceversa, indicating that IL-4 and IL-13 share receptor components. Inaddition, a mutant form of IL-4, Y124D, is capable of inhibiting bothIL-4 and IL-13 biologic responses in lymphoid and nonlymphoid celltypes. The nonlymphoid cell types are predominantly negative for γ_(c),while all of these cell types express varying amounts of IL-13Rα andIL-4Rα. Both IL-4Rα and IL-13Rα, when expressed alone, bind theircognate ligands with a similar high affinity, with K_(a)s ofapproximately 10¹⁰ M⁻¹. The soluble form of IL-4Rα has been shown toretain this ligand-binding ability. Soluble forms of IL-13α haverecently been shown to retain ligand-binding ability both in vitro andin vivo. When IL-4Rα and IL-13Rα are coexpressed, they are capable offorming a complex that can be impacted by both ligands as well asantagonistic ligands like IL-4-Y124D.

[0044] Human IL-13Rα is expressed at either low or moderate levels atboth the mRNA and protein level by a variety of hematopoietic andepithelial cell lines. In colon carcinoma cells, specific signalingevents, namely, Jak 2 tyrosine kinase activation, have been shown tooccur in conjunction with IL-4's biologic effects.

[0045] The cytotoxic activity exhibited by IL-4-Pseudomonas exotoxin(IL-4-PE) and IL-13-PE fusion molecules provides further evidence of theefficient internalization of these receptor systems, since a wide rangeof carcinoma cell lines are quite sensitive of these cytotoxins. Severaldifferent IL-4-PE constructs have been made that are active in vitro andin a mouse xenograft model, and analogous IL-13-PE constructs have beenshown to possess equally potent in vitro activity. Likewise,diphtheria-IL-4 fusion proteins (e.g. DAB₃₈₉IL-4) exhibit in vitrocytotoxic activity.

[0046] IL-13's effects are comparable to IL-4, both in unmodified formand linked to PE. However, unmodified IL-13 can be given at higher dosesthan IL-4 and IL-13-PE shows less toxicity towards hematopoietic cellsthan IL-4-PE. Epithelial cancer cell lines can be targeted and killed byIL-3-toxins as readily as by corresponding IL-4-toxins, indicating thatthe IL-13 receptor complex internalizes as efficiently as the IL-4receptor complex. Unlike IL-4, IL-13 has no biologic effect on T cells.For these reasons, the IL-13 receptor system is preferred to the IL-4receptor system for diagnostic and therapeutic use in vivo.

[0047] In a preferred embodiment, the IL-13 receptor system is used inconjunction with a functional antibody, preferably a single chain mAb(scF_(v)), to carcinoembryonic antigen (CEA). CEA represents anattractive antigenic target for several reasons. It is atumor-associated antigen that it is absent or poorly expressed by normaltissues and highly expressed by the vast majority of carcinomas ofcolon, lung, breast, pancreatic, gastric, ovarian, and medullary thyroidorigin. High incidence and mortality rates for these cancers coupledwith suboptimal diagnostic and therapeutic options result in a seriousand persistent overall public health problem.

[0048] CEA is a glycosylated cell surface protein of approximately 180kDa, and is a solid tumor antigen that has been extensively studiedclinically, both as a circulating tumor marker and as an antigenictarget for radiolabeled mAbs for imaging and therapy. A number ofanti-CEA antibodies have been under study in phase I-III clinicaldiagnostic and therapeutic trials. Exemplary of an anti-CEA mAb is theMN-14mAb. A humanized version of this mAb, hMN-14, in which humanconstant and framework regions replace the corresponding mousesequences, has been constructed and expressed and is the mAb used inthese clinical trials. A ^(99m)Tc-labeled Fab′ fragment of another,related anti-CEA mAb, Immu-4, has received FDA approval for thedetection and staging of colon cancer.

[0049] Though promising as an imaging agent, radiolabeled anti-CEA mAbsin the therapeutic mode previously have yielded few responses. A lowresponse rate resulted even when an anti-CEA mAb was co-administeredwith an anti-TAG-72 mAb, recognizing a second, distinct highly expressedtumor-associated antigen, along with IFN-α, which upregulates bothantigens, on a group of patients with metastatic colon cancer. Advancedcolon cancer likewise has been quite resistant to all chemotherapeuticcombinations tested to date.

[0050] The present invention seeks to overcome these therapeuticbarriers, which are common to most solid tumors, by enhancing theinternalization of CEA. An anti-CEA targeting moiety, such asshIL-13Rα-anti-CEA scF_(v) fusion protein, is administered to a subjectand, after the targeting moiety localizes at the tumor sites, IL-13ligand armed with a therapeutic or diagnostic moiety is delivered. Thissystem provides diagnostic and therapeutic options for the large numberof CEA+ malignancies, including cancers of the lung, colon, breast,stomach, ovary and pancreas, most of which express both CEA and IL-13receptor components. The IL-13 receptor components enable rapidinternalization of the armed ligand, to selectively deliver high levelsof cytotoxic agents to a large group of tumors. Targeting a highlyexpressed antigen, such as CEA, in a sIL-13Rα-anti-CEA scF_(v) fusionmolecule, increases the typically low number of cytokine receptors by upto two orders of magnitude.

[0051] The IL-15/IL-15 receptor system is a good candidate for targetingB-cell and T cell malignancies, normal or activated B cells, andactivated T cells. The IL-15/IL-15 receptor system shares severalfeatures in common with the IL-2/IL-2 receptor system. In vitro and invivo, both IL-2Rα and IL-4Rα have soluble forms, which bind its cognateligand. The IL-2/IL-2 receptor system has a trimeric receptor. IL-15,like IL-2, uses IL-2Rβ and γ_(c), but not IL-2Rα (formerly Tac). Thespecific alpha chain of IL-15 (IL-15Rα) has homology and a similarstructural organization to IL-2Rα. While IL-15Rα is homologousstructurally to IL-2Rα, it has a significantly higher affinity for itscognate ligand, with a K_(a) of ˜10¹⁰ m⁻¹.

[0052] IL-2Rα expressed in the absence of the other two chainsinternalizes slowly, but when juxtaposed to the other subunits by thepresence of ligand, the entire ligand/αβγ complex internalizes at therapid rate intrinsic to the IL-2Rβ/γ_(c) dimer (t_(1/2) of approximately15 minutes). By comparison IL-15Rα has an affinity for its cognateligand (K_(a)≧10¹⁰ M⁻¹) that is at least two orders of magnitude greaterthan that of IL-2Rα for IL-2.

[0053] T, B, NK and monocyte populations respond to IL-15 andcorrespondingly are positive for IL-2Rβ/γ_(c)+/−IL-15Rα. In addition,B-lymphoma/leukemia expresses IL-2Rβ, and γ_(c) is nearly uniformlyexpressed by all normal and malignant hematopoietic cells. The minimalfunctional receptor structure for IL-2 and IL-15 requires co-expressionof IL-2Rβ and γ_(c). Therefore, both normal leukocyte populations, whichmay be pathogenic and which have been targeted in immune/inflammatoryconditions, as well as the malignant counterparts of B-cells and T-cellscan be targeted according to the invention. While similar in manyrespects, the IL-15 receptor system provides at least one advantage overthe IL-2 receptor system, having somewhat less capacity to induceclinically apparent vascular permeability.

[0054] In a preferred embodiment, the IL-2 or IL-15 receptor system isused in conjunction with a functional single chain mAb (scF_(v)) toHLA-DR. HLA-DR is a favorable antigenic target. It is expressed by arange of hematopoietic malignancies (particularly, B-lymphomas andB-leukemias) as well as certain normal immune cells, such as B cells,monocytes, dendritic and activated T cells. Like CEA, it is expressed atlevels that can reach 10⁶ sites/cell, and is internalized at a slowrate.

[0055] The absence of HLA-DR antigen from many critical normal cellpopulations, such as pluripotent bone marrow stem cells, adds to itsclinical utility. This undifferentiated stem cell population canreplenish any depletions in differentiated normal cell populations, ashas been observed in myelotoxic situations such as cancer chemotherapyand bone marrow transplantation. Moreover, in certain severe cases ofimmune-mediated disease, these cell populations may need to beeliminated or significantly attenuated.

[0056] There is a large body of clinical experience in the treatment ofB-cell malignancies with mAbs directed against HLA-DR (using the Lym-1mAb), as well as other B-cell-specific mAbs, including CDs19-22 andCD37. In these studies, radiolabeled mAbs or mAbs linked to toxin havebeen used therapeutically against malignant B cells. Immunotoxins alsohave been employed to eliminate pathogenic T cells in unrelated-donorbone marrow transplantation.

[0057] Effective and selective cellular cytotoxicity via the IL-15receptor and single chain mAb (scF_(v)) to HLA-DR according to theinvention yields significant clinical benefits in a wide rangeofcancers, including B-cell lymphomas and leukemias, as well as manyimmune-mediated diseases including autoimmune and inflammatory diseasessuch as autoimmune diabetic states, inflammatory bowel disease, systemiclupus erythematosus, rheumatoid arthritis, and severe psoriasis, andallograft reactions such as organ transplant rejection,graft-versus-host disease in bone marrow transplantation. Specifictargeting of highly expressed HLA-DR antigen on an activated T cellpopulation with an sIL-15Rα-anti-HLA-DR scF_(v) targeting moietyachieves more specificity and effectiveness in eliminating reactive Tcell populations.

[0058] In accordance with the invention, an anti-HLA-DR targetingmoiety, for example, a shIL-13Rα-anti-HLA-DR scF_(v) fusion protein suchas sIL-15Rα-Lym-2 scF_(v), is administered to a subject. After thetargeting moiety localizes at the therapy site, IL-15 ligand armed witha therapeutic or diagnostic moiety is delivered. The IL-15 receptor ispredicted to enhance the internalization of HLA-DR, to selectivelydeliver high levels of cytotoxic agents. Here again, mAb fragments suchas scF_(v) can be used to provide the added advantages of rapid bloodand organ clearance. By careful selection of components, deliverysystems with minimal immunogenicity can be achieved. The armed ligand,the R-α moiety and the mAb scF_(v) can be tailored to fit thecharacteristics of the particular disease.

[0059] The present invention is designed to provide highertumor/non-tumor ratios, as can be achieved with traditional pretargetingsystems that utilize avidin or streptavidin and biotin, whileeliminating certain problems associated with these systems. While theavidin-biotin system has a very high affinity, clinical experience hasshown that approximately 20-30% of patients mount an antibody responseagainst avidin and up to 70% make antibodies to streptavidin. Thepresent invention avoids the immunogenicity of avidin and biotin. Athree-step approach can be implemented by using an anti-idiotypic mAb,W12, that is reactive to the antigen combining site of MN-14, andthereby any humanized version of MN-14. The W12 mAbs have beengalactosylated, which allows for rapid blood clearance of unlabeledMN-14-based reagent through the hepatic asialoglycoprotein receptor.These pretargeting approaches strive for maximal blood and organclearance of the first step unlabeled pretargeting agent prior toadministering the armed second or third step reagent, in order tominimize normal tissues' exposure to armed diagnostic or therapeuticagents and to maximize tumor/normal tissue ratios of the armed agent.

[0060] In sum, the present invention approach offers potentiallybeneficial alternatives to current approaches for several reasons.First, it uses agents with decreased immunogenicity. mAb fragments haveinherently lower immunogenicity, particularly hMN-14mAb which has beenhumanized. The receptor and ligand components are native andnon-immunogenic. Second, the invention uses a pretargeting strategy thatpermits higher specific delivery of armed ligand to tumor sites orpathogenic cell populations. Third, the invention maximizesinternalization of ligand to allow higher intracellular concentration ofarmed ligand and better diagnostic or therapeutic effects. Fourth, theinvention allows a therapeutic approach with combinations or sequenceswith various agents including drugs, toxins, radionuclides, antisenseand antigene reagents. To accomplish this, different armed forms of thesame ligand are used. Finally, the present invention uses cytokineswhich appear favorable in their toxicity profiles and can be applied toa wide range of diseases.

[0061] The following examples are illustrative of the present invention,but are not to be construed as limiting.

EXAMPLE 1 Construction of a Bifunctional Soluble IL-13Rα-MN-14scF_(v)Fusion Protein

[0062] An MN-14scF_(v) was produced by PCR amplification of cDNA fromthe humanized MN-14transfectoma. The linker used for MN-14scF_(v) was a15-amino acid linker (GGSGS)₃ (SEQ ID NO: 1) and the orientation wasV_(L)-linker-V_(H). After confirmation of the DNA sequences, the singlechain construct was subcloned into an appropriately restrictedcontaining expression plasmid used for other scF_(v)s. This constructthen was subcloned into BL21(λDE3) E. coli for expression.

[0063] The protein was solubilized and renatured from inclusion bodiesand was purified by sequential anion exchange and gel filtrationchromatography. After functional evaluation, the scF_(v) fragment wasligated to a DNA fragment encoding PE40. This immunotoxin was shown tohave specific cytotoxicity for CEA+ cell lines.

[0064] Another single chain construct also was made. This was made withthe opposite 5′-3′ orientation of the heavy and light chains, wasassembled in pCANTABE5E (Pharmacia Biotech, Piscataway, N.J.) andexpressed in phage. Specific binding of recombinant phage expressingthis scF_(v) was demonstrated by ELISA.

[0065] The V_(L)-linker-V_(H) sequence is used for construction of theIL-13Rα-MN-14 fusion protein, as diagrammed below. A 23-amino acidlinker is used between shIL-13Rα and the scF_(v). Kurucz et al. (1995).Alternatively, the (GGSGS)₃ (SEQ ID NO: 1) linker which was used inconstruction of the MN-14 scF_(v) described above is used. Theconfiguration of this fusion protein is:

[0066] shIL-s13Rα-linker—V_(L)-(GGSGS)₃—V_(H)

[0067] The DNA fragment encoding the soluble, extracellular domain ofIL-13Rα is obtained by PCR amplification from positive cell lines,currently Caki-1, HuT 102B2 and A549. PCR primer pairs for RT-PCR aresynthesized, including a primer pair for cloning. The primer pairs spanalmost the entire extracellular domain of the receptor.

[0068] The primers include unique restriction enzyme sites to allow fordirectional cloning into the expression vector, pSinrep 5. This vectoris part of a high-level Sindbis virus, mammalian expression system(Invitrogen, San Diego, Calif.). The recombinant plasmid will includethe wild-type signal sequence and hence can be secreted into the cellculture medium. The ligand-binding region is predicted to lie in theN-terminal half of the molecule, a considerable distance away from thescF_(v) domains, and thus the above configuration was selected. To allowunhindered folding of individual domains a 23-amino acid linker isincluded, as has been described for a bispecific single chain protein,i.e., a fusion of two scF_(v)s.

[0069] In order to retain sequences potentially important forinteractions of IL-13R with partner proteins such as IL-4R, it ispreferable to retain the WSXWS domain and all of the conserved cysteineresidues, along with nearly all of the extracellular domain. Thismaximizes the possibility of interaction with associated proteins in themembrane, thereby facilitating the binding and internalization of theIL-13/IL-13 receptor complex.

EXAMPLE 2 Expression and Purification of the Bifunctional SolubleIL-13Rα-MN-14 scF_(v) Fusion Protein

[0070] Bacterial clones containing recombinant pSinRep5 plasmids arescreened and those with correct sequences are used for expression.Recombinant virions are produced to provide higher expression and astable reusable stock for multiple transductions. This is accomplishedby co-transfection of in vitro transcribed RNA from the recombinantplasmid plus a replication-deficient helper virus DNA template (as permanufacturer's instructions). Alternatively, an E. coli-based expressionsystem, similar to the one used for production of MN-14scF_(v) is used.

[0071] When recombinant Sindbis virions are used, RNA transcription isperformed from the recombinant plasmid (prepared from the initialpSinRep5 plasmids) and a helper virus plasmid that is included in astandard kit. (Invitrogen, Inc., San Diego, Calif.) RNA yields areassessed by agarose gel electrophoresis and the RNAs then are used toco-transfect the BHK cell line. This is done using cationic liposomesand/or electroporation. After 3 days in culture the supernatant from thetransfection is collected and used to transduce fresh BHK cells toassess viral titer and to assess the level of recombinant proteinexpression. For all transductions, cells are plated initially inFCS-containing medium. After approximately 20 hours the medium ischanged to serum-free medium. After 3 days, supernatants fromrecombinant and non-recombinant controls are collected and aliquots areconcentrated by centrifugal ultrafiltration for total proteindetermination (Coomassie Plus™, Pierce, Rockford, Ill.).

[0072] Aliquots of the concentrated supernatant are fractionated bySDS-PAGE. The gel is divided for Coomassie staining and electroblottingonto PVDF for Western blotting. sIL-13Rα-MN-14 scF_(v) expression isdetected with goat anti-human IgG-peroxidase to detect Hmn-14 sequencesand development by chemiluminescence. After exposure to photographicfilm, the blot is examined for a specifically stained band ofapproximately 70 kDa.

[0073] Once expression is confirmed the transductions are scaled up. Forlarge-scale expression runs, the purification scheme includes anionexchange, gel filtration and/or other HPLC modes with determination offinal recovery and purity. In initial genetic constructs a C-terminalhexahistidine or related inert affinity tag sequence will be added toassist purification.

EXAMPLE 3 Assay of Antigen and Ligand Binding Activity of SolubleIL-13Rα-MN-14 scF_(v) Fusion Protein

[0074] Purified sIL-13Rα-MN-14 scF_(v) is ¹²⁵I-labeled by the lodogen(Pierce) method to approximately 5 μCi/μg. The LS174T cell line is usedfor binding studies since it expresses high levels of CEA and has low tomoderate IL-13 binding.

[0075] An amount of 1×10⁶ washed LS 174T cells/tube is suspended in 100μl of binding buffer (RPMI 1640/10% FCS). Either labeled sIL-13Rα-MN-14sc_(v) at 10 nM alone or labeled sIL-13Rα-MN-14 scF_(v) at 10 nMtogether with a 200 fold molar excess of the unlabeled protein is addedto replicate tubes. The overall ability to bind antigen is assessed byusing tracer concentrations (50-200 pM) of labeled fusion protein andlarger numbers of LS174T cells. The bindable fraction should be greaterthan 50% in antigen excess.

[0076] IL-13 binding ability is assessed using ¹²⁵I-IL-13 (Iodogenlabeled as before). To replicate tubes containing 1×10⁶ LS174T cells isadded either unlabeled fusion protein or buffer. After 30-40 min at 4°C. cells are washed and ¹²⁵I-IL-13 is added, either in the presence orabsence of cold IL-13. A significant positive increment in specificIL-13 binding indicates a functional IL-13Rα moiety.

EXAMPLE 4 Assay of Ability of Soluble IL-13Rα-MN-14 scF_(v) FusionProtein to Internalize CEA

[0077] sIL-13Rα-MN-14 scF_(v) is radioiodinated to a specific activityof approximately 5-10 μCi/μg. Ten nM ligand is added to replicate tubesof 2.5×10⁵ LS174T cells/tube, and incubated at 4° C. for one hour. Cellsare washed twice with binding buffer and plated in 24 well plates. Tosome tubes a 200-fold molar excess of cold ligand is added to assessspecific binding. To other tubes, 1 nM unlabeled rhIL-13 is added toassess effects on internalization and processing.

[0078] Supernatants are removed at multiple time points and brought to10% TCA to precipitate intact the label, so as to distinguish labelwhich dissociates versus label that is internalized, catabolized andreleased. Plates are washed three times with binding buffer. Cells aresolubilized with 0.4 ml 2 N NaOH for counting.

EXAMPLE 5 Biodistribution of Radiolabeled IL-13

[0079] Biodistribution of IL-13 is studied in CEA+ tumor-bearing nudemice, using the LS174T model system. Five week old female nude mice areinjected with 5×10⁶ cells from the LS1747T or HT-29 cell lines, both ofwhich are colon cancer cell line, resulting in development ofsubcutaneous tumors. Biodistribution of ¹²⁵I-IL-13 in micesimultaneously injected with ¹³¹I-labeled MN-14Fab′ or MN-14scF_(v)having similar K_(d)s and MWs is compared.

[0080] Dual label biodistribution experiments using radioiodinated IL-13with IL-4 and MN-14 Fab′ as controls have been performed in both theLS174T and HT-29 tumor xenograft model systems to assess tumor andnormal organ uptake of these agents. Uptake values observed wereconsistent with both the molecular mass of the agents and the level ofexpression of the corresponding binding proteins in the tumor (cognatereceptor proteins for the cytokines and CEA for MN-14Fab′). In summary,IL-13 uptake in these colon tumors was typically in the range of 0.2-1.0percent of injected dose/gram tissue (%ID/g), while the correspondinguptake values for MN-14Fab′ were 4.0-7.5 for HT-29 and 7.0 for >20 forthe LS174T tumors. The IL-4 control behaved similarly IL-13. Theseresults are consistent with the low levels of cognate cytokine receptorin both of these tumors (as assessed by radiotracer binding to harvestedtumor cells), the intermediate levels of CEA on HT-29 and the highlevels of CEA on LS 174T. Tumor uptake values were maximal for bothcytokines and Fab′ at 5 hours post-injection, which is consistent withtheir rapid clearance that is a consequence of their low molecularmasses. Renal uptake of all agents was high and also peaked at 1-5hours, which is consistent with the known renal clearance mechanism ofsuch agents. Liver showed the highest uptake of IL-14 and IL-14 amongstother normal organs, which is consistent with the known preclinical andclinical hepatotoxicity seen with higher doses of these agents.

[0081] A parallel set of experiments using the Ramos B cell tumorxenograft model were performed using radioiodinated IL-15, with IL-2 ascytokine control and LL2 FAB′ (recognizing the CD22 B cell antigen) astheir control. Overall results were similar in this model system, exceptthat liver intake vales were lower with these cytokines compared toIL-13 and IL-14. These experiments confirmed the expectedpharmacokinetic and pharmacodynamic behavior of the IL-4, IL-13, IL-2and IL-15 cytokine agents. The results indicate that tumor uptakereflects the low basal level of expression of the cognate cytokinereceptors in both CEA+ carcinomas as well as lymphomas, which can beincreased by first targeting tumors with antibody-Rα fusion proteins.Once targeted with this bifunctional agent, administration of armed,cognate cytokine is predicted to result in higher uptake than wouldoccur with either the antibody or cytokine without pretargeting with theantibody-Rα agent.

EXAMPLE 6 Construction of a IL-13/Onconase Immunotoxin

[0082] A fusion protein consisting of IL-13 and onconase is geneticallyengineered following procedures outlined by Rybak for the production ofmAb-onconase fusion proteins. Tumor Targeting 1:141-147 (1995). Briefly,a sequence-confirmed fragment corresponding to the mature IL-13 proteinis ligated to the sequence of onconase with the IL-13 sequence lying 5′,though the other orientation also can be evaluated. Onconase genes arecloned from two or more frog species. Authentic fragments representingthe fusion sequence are subcloned into the pET21d vector again using aC-terminal hexahistidine tag. The complete sequence encoding the entireIL-13-onconase fusion protein is confirmed in the pET vector in the XL1Blue strain as above. Appropriate clones are expanded to produce plasmidfor transformation of the AD494 (DE3) E. coli expression strain.

[0083] Transformed clones are picked and grown in small scale culture,induced with IPTG, lysed in SDS sample buffer and separated on aSDS-PAGE gel for Coomassie staining and transblotting for detection bothwith anti-IL-13 antibodies and anti-onconase antibodies. Isolation andwashing of inclusion bodies, their solubilization, renaturation andsubsequent purification is performed using the steps outlined above. Thefinal product is tested for its ability to bind the IL-13 receptor bylabeling with ¹²⁵I and comparing it with equimolar concentrations ofsimilarly labeled IL-13 in the cell binding assay described above.Conjugates with the ability to bind IL-13 are tested for cytotoxicity oncell lines known to express receptors for IL-13.

[0084] The conjugate is tested in the presence and absence of thesIL-13Rα-hMN-14scF_(v) fusion protein to determine toxicity and theability to bind and internalize greater amounts of the immunotoxin. Adose response curve for each experimental and control condition isgenerated.

EXAMPLE 7 Therapy of Colon, Lung, Breast, Pancreatic, Gastric, Ovarianand Medullary Thyroid Carcinoma

[0085] A patient having carcinoma of the colon, lung, pancreas, stomach,ovary, breast or medullary thyroid is infused intravenously with asterile, pyrogen-free solution containing sIL-13Rα-hMN-14scF_(v) fusionprotein in phosphate-buffered saline (PBS), prepared according toExamples 1 and 2. Time is allowed for the fusion protein to bind tomalignant tumor cells and to clear substantially from the circulation ofthe patient.

[0086] The patient then is infused intravenously on pre-determinedschedule with a sterile, pyrogen-free solution that containsradioisotopically-labled or drug-conjugated IL-13 or IL-13/onconaseimmunotoxin conjugate, prepared according to Example 6. Subsequentradiologic or radioimmunodetection methods are then used to evaluateantitumor responses.

[0087] While the invention has been described in detail with respect toparticular preferred embodiments, it should be understood that suchdescription is presented by way of illustration and not limitation. Manychanges and modifications within the scope of the present invention maybe made without departing from the spirit thereof, and the inventionincludes all such modifications. For example, sIL-15Rα-Lym-2 scF_(v)fusion protein can be constructed, expressed, purified and evaluatedusing similar methodology to that described for solubleIL-13Rα-MN-14scF_(v) fusion protein in Examples 1 through 3. The cognateligand for receptor moiety, IL-15, can be similarly armed or labeledwith drug, toxin or radionuclide. In this case, the B-cell lines Ramosand RL can be used as the in vitro and preclinical mouse tumor xenograftmodel. The ultimate clinical malignancies that would be addressed are Band T cell lymphoma/leukemia, Hodgkin's disease and other HLA-DR+cancers. This approach can also be used to suppress HLA-DR+ cellpopulations, which mediate immunologic or inflammatory diseases.

1 1 1 15 PRT Artificial Sequence Description of Artificial SequenceLinker 1 Gly Gly Ser Gly Ser Gly Gly Ser Gly Ser Gly Gly Ser Gly Ser 1 510 15

What is claimed is:
 1. A targeting moiety comprising a conjugate of anantibody linked to a ligand-binding region of a receptor subunitselected from the group consisting of interleukin-2 receptor α(IL-2α),interleukin-4 receptor α(IL-4α), and interleukin-15 receptor α(IL-15Rα),which antibody is specific for a cell marker specific to a targetedcell.
 2. A targeting moiety as claimed in claim 1, comprising a covalentconjugate in which the antibody is covalently linked to theligand-binding region of the receptor.
 3. A targeting moiety as claimedin claim 1, comprising a fusion protein of the antibody and theligand-binding region.
 4. A targeting moiety as claimed in claim 1,comprising a bispecific antibody that has a first specificity for a cellmarker specific to a targeted cell and a second specificity for theligand-binding region.
 5. A targeting moiety as claimed in claim 1,wherein the antibody is specific to CEA.
 6. A targeting moiety asclaimed in claim 1, wherein the antibody is specific to HLA-DR.
 7. Atargeting moiety as claimed in claim 1, wherein the receptor subunit isIL-4α.
 8. A targeting moiety as claimed in claim 1, wherein the receptorsubunit is IL-15Rα.
 9. A targeting moiety as claimed in claim 1, whereinthe antibody is specific to HLA-DR and is linked to the ligand-bindingregion of IL-15Rα.
 10. A targeting moiety as claimed in claim 1, whereinthe antibody is specific to CEA and is linked to the ligand-bindingregion of IL-15Rα.
 11. A targeting moiety as claimed in claim 1, whereinthe antibody is specific to HLA-DR and is linked to the ligand-bindingregion of IL-4Rα.
 12. A targeting moiety as claimed in claim 1, whereinthe antibody is specific to CEA and is linked to the ligand-bindingregion of IL-4Rα.
 13. A targeting moiety as claimed in claim 1, whereinthe antibody is specific for a cell marker specific to a B-cell.
 14. Atargeting moiety as claimed in claim 1, wherein the antibody is specificfor a cell marker specific to a malignant B-cell.
 15. A targeting moietyas claimed in claim 1, wherein the antibody is specific for a cellmarker specific to an activated B-cell.
 16. A targeting moiety asclaimed in claim 1, wherein the antibody is specific for a cell markerspecific to a normal B-cell.
 17. A targeting moiety as claimed in claim1, wherein the antibody is specific for a cell marker specific to aT-cell.
 18. A targeting moiety as claimed in claim 1, wherein theantibody is specific for a cell marker specific to a malignant T-cell.19. A targeting moiety as claimed in claim 1, wherein the antibody isspecific for a cell marker specific to an activated T-cell.
 20. Acomposition comprising a targeting moiety according to claim 1, and apharmaceutically acceptable carrier.
 21. A composition comprising atargeting moiety according to claim 7, and a pharmaceutically acceptablecarrier.
 22. A composition comprising a targeting moiety according toclaim 8, and a pharmaceutically acceptable carrier.
 23. A kit comprisinga conjugate of IL-15 linked to a drug, radionuclide or toxin, and atargeting moiety comprising an antibody specific for a cell markerspecific to a targeted cell, linked to the ligand-binding region ofIL-15Rα.
 24. A kit as claimed in claim 23, wherein the antibody isspecific for a cell marker specific to a B-cell.
 25. A kit as claimed inclaim 23, wherein the antibody is specific for a cell marker specific toa malignant B-cell.
 26. A kit as claimed in claim 23, wherein theantibody is specific for a cell marker specific to an activated B-cell.27. A kit as claimed in claim 23, wherein the antibody is specific for acell marker specific to a normal B-cell.
 28. A kit as claimed in claim23, wherein the antibody is specific for a cell marker specific to aT-cell.
 29. A kit as claimed in claim 23, wherein the antibody isspecific for a cell marker specific to a malignant T-cell.
 30. A methodof treatment for cancer or an immunologically-mediated or infectiousdisease, comprising: first administering to a subject in need of suchtreatment a targeting moiety comprising a antibody specific for a cellmarker specific to a targeted cell, linked to the ligand-binding regionof IL-15Rα, and then administering to the subject a therapeuticallyeffective amount of a conjugate of IL-15 linked to a drug, radionuclideor toxin.
 31. A method as claimed in claim 30, wherein the antibody isspecific for a cell marker specific to a B-cell.
 32. A method as claimedin claim 30, wherein the antibody is specific for a cell marker specificto a malignant B-cell.
 33. A method as claimed in claim 30, wherein theantibody is specific for a cell marker specific to an activated B-cell.34. A method as claimed in claim 30, wherein the antibody is specificfor a cell marker specific to a normal B-cell.
 35. A method as claimedin claim 30, wherein the antibody is specific for a cell marker specificto a T-cell.
 36. A method as claimed in claim 30, wherein the antibodyis specific for a cell marker specific to a malignant T-cell.
 37. Atargeting moiety comprising a conjugate of an antibody linked to aligand-binding region of interleukin-13 receptor α(IL-13Rα), whichantibody is specific to HLA-DR.
 38. A composition comprising a targetingmoiety according to claim 18, and a pharmaceutically acceptable carrier.